The recognition of antigen by a T cell through its T cell receptor (TCR) is a seminal event in the induction of an adaptive immune response. The TCR recognizes a foreign peptide/MHC complex on the surface of an antigen presenting cell; however, self-peptide/MHC complexes also play a critical role. These self-pMHC interactions are needed for positive selection of T cells in the thymus to generate a MHC restricted repertoire and negative selection to purge the repertoire of self-reactive T cells. In peripheral T cells TCR:self-pMHC interactions are critical for survival, for setting signaling thresholds, and for responses to foreign antigens. The TCR recognition of self-pMHC complexes is weak in nature, but still results in signaling events in the T cell. An APC such as a dendritic cell expresses thousands of different self-pMHC complexes on its surface, complicating the investigation of the nature of the signals stimulated in the T cell. Our novel approaches leverage TCRs with distinct affinities for a self-pMHC, which will allow us to identify a self-pMHC that augments T cell motility and responses in vivo. Specifically, we have generated two TCR transgenic mice, LLO56 and LLO118, which both recognize the immunodominant Listeria epitope (LLO). The advantage of studying these two T cells is that they have distinct in vivo behaviors in response to Listeria infection. Moreover, and a central point for this application, is that the intrinsic sensitivity of LLO118 and LLO56 is set and maintained by self pMHC, with the LLO56 T cells having stronger self-pMHC interactions. Our initial in vivo experiments indicate marked differences in their transit times in lymphoid tissues, pointing to the recognition of self peptides as a major determinant of T cell motilities. The hypothesis this proposal tests is that there is a set of self-pMHC displayed on an APC, which make specific, but weak, interactions with LLO56 and LLO118 T cells in vivo. These interactions set the signaling threshold and are essential for T cell function and survival. To test this hypothesis, two specific aims are proposed. In aim 1, we propose to Identify self-pMHCs that potentiate T:DC interactions in the absence of antigen. We have developed a novel mini-self-pMHC repertoire system in which we can express 3 to 9 defined covalently linkered self-pMHC as the only class II molecules on a DC. We will test a series (up to 30) of self-pMHC in vivo using 2-photon microscopy for their abilities to slow the motility of LLO56 or LLO118 T cells. In Aim 2, we will determine the specificity and in vivo functional consequences of self-pMHC:T cell interactions. The active self-pMHC identified in Aim 1, will be analyzed for how specifically they are recognized by the T cells. The active self-pMHC will then be studied for their ability to augment an in vivo response to foreign antigen. By identifying an active self-pMHC, in future studies using this defined system, the signaling pathways in T cells induced by these self-pMHC can be elucidated. Knowing these pathways and how they are activated, could lead to the development of pharmacological enhancements for T cell survival and function.